ABSTRACT Botanical natural products are consumed every day by Americans either through their regular diet or via dietary supplements. In particular, herbal dietary supplements are consumed by 18-30% of the U.S. population, with over $8 billion in annual sales. Because consumers often incorrectly believe all dietary supplements are harmless, they take dietary supplements in addition to medically prescribed pharmaceuticals and often do not disclose this information to their physicians. However, recent studies show that at least two out of the 40 top selling dietary supplements are not safe to take with some pharmaceuticals, which has led to concerns about potential interactions with other natural products. One of the most common herb-drug interactions involves the induction/inhibition of the cytochrome P450 enzymes (CYP), a superfamily of enzymes found mainly in the liver and involved in the metabolism of a plethora of xenobiotics. Common herbal medicines such as St. John?s wort, garlic, black pepper (piperine), ginseng, and gingko, have given rise to serious clinical interactions when co-administered with prescription medicines. There is an urgent need to evaluate natural product-drug interactions using well-validated methodology at a large scale. We propose to screen a large one-of-a-kind library (Quave Natural Products Library, QNPL) of >1,900 botanical extracts for their potential interference with drug metabolizing enzymes (CYPs). The composition of this library is innovative and includes extracts derived from dietary botanicals used in traditional medicine, herbal teas, and foods from diverse geographic regions. In Aim 1, we will create extracts of the top 40 herbs of commerce and known botanical CYP inhibitors, and then screen these 40 herbs and the QNPL collection of >600 botanicals (>1,900 extracts) against eight cytochrome P450 enzymes with an in vitro model recommended by current FDA guidance. We will prioritize the most potent extracts identified as CYP inhibitors for follow up studies by performing a dose-response analysis. In Aim 2, we will isolate and elucidate structures of active compounds from botanicals exhibiting potent activity. To identify any known compounds prior to this work, we will first dereplicate the prioritized extracts using targeted and untargeted LC-MS metabolomics. Then, hit extracts will undergo bioactivity fractionation to determine the specific compounds contributing to an extract?s activity. Lastly, we will use MS, NMR, and X-ray crystallography to determine the identity and structure of the bioactive compounds. This study is closely aligned with the RFA-AT- 20-001 ?Preclinical Screening for Natural Product Drug Interactions? objectives to 1) Screen large libraries of natural products relevant to human consumption (e.g. herbals, botanicals, foods, dietary supplements, phytochemicals) in well validated assays of Phase I and Phase II drug metabolizing enzymes and drug transporters; and 2) Elucidate active compounds in complex mixtures that have demonstrated potent activity in assays relevant to human drug metabolism.